Content of industrially produced trans fatty acids in breast milk: An observational study

Abstract Breast milk may contain industrially produced trans fatty acids (TFAs), which can affect the content of essential fatty acids (EFAs). This could have significant implications for the child's development. The fatty acids present in breast milk can be modified by adjusting the mother's diet. The objective of this study was to determine the content of industrially produced TFAs present in colostrum, transitional milk, and mature milk produced by mothers between 18 and 45 years of age in the state of Querétaro, Mexico, based on a longitudinal observational study. The TFA content in the breast milk of 33 lactating women was analyzed using gas chromatography. The mothers’ consumption of TFAs was also estimated by analyzing a log prepared through 24‐hr dietary recall (24HR) obtained in each period. The TFA content in the mothers’ diet was similar across the colostrum, transitional milk, and mature milk phases: 1.64 ± 1.25 g, 1.39 ± 1.01, and 1.66 ± 1.13 g, respectively. The total TFA content was 1.529% ± 1.648% for colostrum; 0.748% ± 1.033% for transitional milk and 0.945% ± 1.368% for mature milk. Elaidic acid was the TFA in the highest concentration in all three types of milk. No correlation was found between the content of industrially produced TFAs in breast milk and the anthropometric measurements of the mother or between the estimated consumption of TFAs and the content of TFAs in breast milk. Elaidic acid and total content of TFAs were negatively correlated (p < .05) with the content of docosahexaenoic acid (DHA) (0.394 ± 0.247) (R = −0.382) in colostrum. The concentration of TFAs was found to correlate with the composition of EFAs in milk.


| INTRODUC TI ON
Breast milk is the ideal food for the newborn (Andreas et al., 2015).
Its nutrients and components are essential for promoting proper growth and development of the infant (Andreas et al., 2015;German & Dillard, 2006;Koletzko et al., 2011). The composition of breast milk is variable and may change according to dietary, genetic, sociodemographic, health, and environmental factors (Burianova et al., 2019;Miliku et al., 2019). It also varies over the course of the postpartum period, when different types of milk are produced in succession. These can be classified into three stages of production: colostrum, transitional milk, and mature milk.
The fat present in breast milk is the infant's main source of energy (50% of total energy) (Marín et al., 2009;Vásquez-Garibay, 2016;Wan et al., 2010). This fat consists primarily of saturated (42%-47%) and unsaturated (53%-58%) fatty acids (Macías et al., 2006;Marín et al., 2009). While 60%-70% of the fat in milk is the result of tissue synthesis and maternal fat deposits, approximately 30% of the fat in breast milk is derived from her diet (De Souza Santos da Costa et al., 2016;Deng et al., 2018). The concentration of fatty acid in breast milk depends on the mother's intake of lipids and is therefore linked to the type of diet prevalent in the region where she lives (Deng et al., 2018;Gómez-Cortés & De La Fuente, 2017;Marín et al., 2009;Mazzocchi et al., 2018;Perrin et al., 2018;Samur et al., 2009;Wan et al., 2010). The essential fatty acids (EFAs) contained in breast milk are precursors of long-chain polyunsaturated fatty acids (LC-PUFAs) and play a key role in the growth and maturation of the central nervous system and development of the infant's visual-sensory system. Alterations in its concentration may cause damage to the newborn's cognitive and visual development, causing dysfunction and permanent negative effects on IQ and visual acuity (Birch et al., 2005;De Souza Santos da Costa et al., 2016;Deng et al., 2018;Gómez-Cortés & De La Fuente, 2017;Jensen et al., 2010;Nishimura et al., 2014;Spector & Kim, 2015;Vega et al., 2012). Human milk may contain trans fatty acids (TFAs), which are unsaturated fatty acids with at least one double bond in the trans configuration (Fernández-Michel et al., 2008;Islam et al., 2019;Wu et al., 2017). A TFA concentration of 2%-5% has been reported in human milk (Daud et al., 2013;Krešić et al., 2013;Larqué et al., 2001;Samur et al., 2009;Valenzuela, 2008). The human body does not synthesize trans fats, so their presence in breast milk is related solely to maternal intake (Samur et al., 2009). These TFAs may be either naturally or industrially produced. It is estimated that only 5% of total trans fats in the human diet are natural in origin (Ballesteros-Vásquez et al., 2012). Recent studies have found benefits for the organism from consuming natural TFAs (Kuhnt et al., 2015), while consumption of industrially produced TFAs has been found to have negative health implications (Ginter & Simko, 2016;Islam et al., 2019;Sauvat et al., 2018). Industrially produced TFAs are created through the hydrogenation of vegetable oils in industrial food processing. This is done to improve stability and lengthen shelf life, reduce the cost of these products, and produce desirable organoleptic characteristics for the consumer (Ballesteros-Vásquez et al., 2012;Castro-Martínez et al., 2010;Hyseni et al., 2017). The predominant industrially produced TFA in the food supply is elaidic acid (De Souza et al., 2015;Hauff & Vetter, 2009;Krešić et al., 2013;Villalpando, 2007), which has been found in greater proportion in breast milk (Duran & Masson, 2010). However, other industrially produced TFAs have also been identified in breast milk, such as li-  (Perrin et al., 2018). There is evidence that industrially produced TFAs interfere with the organism's ability to metabolize EFAs, because they compete for the same enzymes that desaturate them (∆5 and ∆6 desaturases), blocking the activity of these enzymes in synthesizing LC-PUFAs, which may have significant implications in newborns (Krešić et al., 2013;Kummerow, 2009;Villalpando, 2007

| Participants
The sample consisted of lactating women who had given birth at the Hospital for Specialties of Children and Women. Participants were recruited in the short-term Postnatal and Gynecology areas.
The inclusion criteria were: clinically healthy women who had given birth to a single child at most 5 days prior to recruitment; who were breastfeeding; aged between 18 and 45 years; and who agreed to participate in the study. The sample excluded mothers who had undergone some kind of drug therapy in the previous 7 days or hormonal therapy in the previous three weeks; those who reported consuming some psychoactive drug during pregnancy or lactation; those who reported some impediment to breastfeeding; those who consumed any nutritional or dietary supplement that contained fatty acids during the lactation period, or those who declined to continue in the study. Initially, 56 women were included in the study, 33 of whom completed the three milk samples.

| Study design and procedures
Each mother was visited three times. During these sessions, anthropometric measurements were taken, and samples of breast milk collected.
The first session was carried out during the colostrum production period (days 1-5 after delivery); the second during the production of transitional milk (days 6-15 after delivery) and the third during the production of mature milk (from day 15 to the end of the first month postpartum).
In the first session, after informed consent was obtained, the birth weight of the newborn was taken from the mother's file and the following questionnaires were applied to the mother: medical history, which included general information and personal pathological, hereditary-family, and gynecological/obstetrical histories; socioeconomic study questionnaire; food surveys; and the infant questionnaire, through which the data on the lactation pattern and the health status of the infant were recorded. In the remaining two sessions, the infant questionnaire and feeding surveys were also applied.

| Collection of breast milk samples
Breast milk was expressed manually by the mother based on the Official Mexican Standard NOM-043-SSA2-2012, Basic Health Services: Promotion and education for health in alimentary matters, published by the Mexican Ministry of Health (Secretaría de Salud, 2013), which provides criteria for guidance and assistance if needed. Colostrum extraction was carried out at the Hospital for Specialties of Children and Women, and the transitional milk and mature milk were extracted at the nursing mother's home.
Between 2 and 5 ml of colostrum was collected in each extraction, and from 10 to 30 ml of transitional milk and mature milk.
The extraction was carried out between 8:00 a.m. and 5:00 p.m. and was obtained in all cases from a single breast.
Once the sample was collected, the milk was labeled with the mother's name, date of extraction, weeks of gestation of the baby, time of birth of the baby, and volume of milk provided. It was then placed in a cooler with prefrozen gel and transported to the Cell Biology and Human Nutrition laboratories of the Faculty of Natural Sciences, UAQ, Juriquilla campus, where it was stored in a freezer at −80°C for subsequent analysis.

| Food surveys and analysis of the maternal diet
The mother's diet was evaluated by analyzing the 24-hr dietary recall (24HR), a structured interview used to capture detailed information on all the foods and beverages consumed in the past 24 hr.
During each visit to the mother, three such records were collected: two pertaining to weekdays and one to a weekend day. This means that nine 24-hr logs were collected for each mother: three for the colostrum period, three for the transitional milk period, and three for the mature milk period. The "Food Base of Mexico: Compilation of the composition of foods frequently consumed in the country"  (Villalpando, 2007). The TFAs present in the mother's diet were quantified and averaged, thus obtaining an estimate of their habitual consumption at each stage of lactation. TFA consumption was expressed in grams and as a percentage of the total caloric value of the diet. The number of kilocalories supplied by TFA per gram was determined, as well as the amount of energy from all the macronutrients consumed, and consumption of TFA was thus calculated as a percentage of the total.

| Obtaining anthropometric measurements from the mothers
During the first visit, the mother's height, prepregnancy weight, and maximum weight during pregnancy were obtained from the clinical record. Subsequently, measurements of weight and body fat percentage were taken from the mothers during their hospital stay (first 5 days postpartum: colostrum stage) and at home (day 5 to 15 postpartum: transitional stage; and >15 days postpartum: mature milk stage). An Omron scale and body composition monitor was used, which functions using bioelectrical impedance analysis (OMRON HEALTHCARE INC, 2017). For measurement, the scale was placed on a flat surface and mothers were asked to take off their shoes and remove any heavy clothing before being weighed. The weight was taken in an upright position, with the feet on the electrodes and the arms hanging parallel to the body. The fat percentage was measured in an upright position, with the feet and hands on the electrodes.

| Fatty acid determinations
The extraction of samples from breast milk for the subsequent determination of TFAs and EFA was carried out using the methodology described by Chávez-Servín, 2009(Chávez-Servín et al., 2009 in the laboratories of the Faculty of Natural Sciences, UAQ. Briefly, the breast milk samples were homogenized, starting with 200 µl of breast milk in each tube; 40 µl of the C13: 0 internal standard solution previously diluted in 1 mg/ml n-hexane was added to each tube; 1 ml of a sodium methylate solution (CH3ONa) in methanol was added; and after shaking, the test tubes were placed in a multiblock heater preheated to 115°C for 17 min; then subsequently cooled in a water bath. Once at room temperature, 1 ml of boron trifluoride in methanol (BF3/MeOH) (14% w/v) was added to each tube; after stirring, the tubes were placed again in the multiblock heater preheated to 115°C for another 17 min; then they were cooled again to room temperature. Subsequently, they were mixed with 2 ml of hexane and 2 ml of saturated NaCl solution. Subsequently, after centrifugation, 1.5 ml of the hexane phase was transferred to Eppendorf tubes in which a spatula tip with anhydrous sodium sulfate was previously added. After stirring and subsequent resting, the hexane phase was transferred to a vial and stored in the freezer at −20°C until the time of injection into the gas chromatograph.
After the samples were processed, the fatty acids were determined at CICATA using an Agilent 6890A gas chromatograph (Agilent Technologies) equipped with a fused silica capillary column SP2560; 100 m × 0.25 mm diameter with 0.2µm film thickness (Supelco, Inc., Bellefonte, PA, USA) and a FID.

| Statistical analysis
A descriptive analysis of the population characteristics was carried out to determine the mean, standard deviation, and minimum and maximum values. A comparative analysis of the means of the anthropometric indicators for each mother was carried out at each of the three milk production stages: maternal weight (kg), body fat (%), body mass index (BMI; kg/m 2 ), weight lost (%) after delivery, and percentage of prepregnancy weight (%). Likewise, the means of the food variables were compared: energy consumption (Kcal), lipids (%), proteins (%), carbohydrates (%), and industrially produced TFAs in the diet (g, %), by a general linear model. A comparative analysis of the composition of fatty acids in colostrum, transitional milk, and mature milk was carried out, using the Friedman test to compare the corresponding means. The population was classified according to the BMI and percentage of body fat. To compare the content of industrially produced TFAs and EFAs in breast milk according to these two classifications, the Kruskal-Wallis test was used. Since most of the TFA and EFA variables did not conform to a normal distribution, in order to measure the association between "EFA and industrially produced TFAs in breast milk" and "industrially produced TFAs in maternal diet," several logistic regressions were performed to evaluate the probability of finding TFA or EFA content above the median value in breast milk, when the mother consumes more calories or a higher percentage of each macronutrient. Finally, the association between the "industrially produced TFAs" and "EFA" variables in breast milk was analyzed for colostrum, transitional milk, and mature milk, analyzing R, R 2 . The confidence interval (CI) for all analyses was 95%, with a statistical significance of p < .05.

| Sample characteristics
The study involved 56 breastfeeding women. In the end, 33 of these mothers completed the three milk samples and the requested questionnaires, and these constituted the sample used. The mean age of the 33 women was 25.3 ± 6.7 years. They were recruited on their first postpartum day, with an average of 38.8 ± 1.1 weeks of gestation, with an average of 2 children (range 1-5). In relation to marital status, 66.6% of participants were living in a domestic partnership, 18.1% were married, and 15.1% were single. Regarding occupation, participants were employed primarily in home and family care (75.75%), working for others (9.09%), students (6.06%), self-employed merchants (6.06%), or professionals (3.03%). The birth route of the most recent child was vaginal delivery in 81.81% of participants, and caesarean section in 18.18%. With regard to support for breastfeeding in the hospital, in the first feeding immediately after birth, 84.84% of the infants were breastfed, 3.03% (one infant) initially received formula (and subsequently received breast milk), and 12.12% received mixed feeding (alternating breast milk and formula). As many as 54.54% of mothers fed their babies breast milk within the first hour of birth; most of the mothers were able to be with their baby during their hospital stay (96.96%); while only one mother (3.03%) was separated from her baby due to health conditions. Finally, the socioeconomic level of mothers was middleto very low-income for more than 90% of the women studied. The largest percentage of participants (60.60%) was in the middle-lower income bracket.

| Anthropometric measurements of mother and infant
The infants involved in this study were generally of normal weight (2500-4500 g) at birth, with a mean of 3025.1 ± 462.6 g. However, 12.1% of infants had low birth weight (<2500 g), according to WHO criteria. None of the newborns weighed more than 4500 g at birth.
Maternal height (cm) was 156.4 ± 5.5. The average weight and pregestational BMI were 58.7 ± 12.8 kg and 23.9 ± 4.8 kg/m 2 , respectively. Maximum weight and weight gain in pregnancy were Weight, BMI, and percentage of prepregnancy weight were all found in higher ranges during the colostrum stage, which is due to weight gain during pregnancy. However, these values decreased progressively and significantly over the course of the colostrum, transitional milk, and mature milk stages (p < .05). These results may be due to the mother's postpartum recovery and the added energy expenditure that breastfeeding implies, which contributes to the return to prepregnancy weight. A significant increase in the percentage of body fat was observed at 5 to 15 days postpartum (transitional stage) compared to the percentage in the colostrum stage. In the colostrum stage, the percentage of mothers with a high percentage of body fat (body fat > 33%) (Gallagher et al., 2000) was lower (39.39%) than in the transitional (48.48%) and mature (54.54%) milk stages. This is probably because the body requires more fat for milk production at each stage ( Table 1).

| Estimation of TFA consumption by nursing mothers
The diet of the 33 women during the three stages of lactation was evaluated. The amount of industrially produced TFAs was estimated, as well as the energy consumption and proportion of fats, proteins, and carbohydrates in their diets ( Table 2).
The average daily consumption of TFAs was 1.56 ± 0.75 g/day, and it accounted for 0.65% ± 0.30% of the total caloric value of the diet. No differences were found in energy intake or in the consumption of lipids, carbohydrates, or TFAs in the three stages. This may be due to the temporal proximity between one evaluation and another (evaluations were made at 1-5 days, at 6-15 days, and at 16-30 days). However, protein consumption was significantly higher The intake of TFAs in the Mexican study population was 1.56 g/day (0.66%), lower than that reported by women in Turkey (2.16 g/day) (Samur et al., 2009) (Daud et al., 2013), China (0.16%-0.34%) (Deng et al., 2018), and Canada (0.8 g/day) (Ratnayake et al., 2014).

| Fatty acid content in breast milk
Fatty acid content in breast milk was analyzed in three postpartum lactation stages (colostrum, transitional milk, and mature milk), including EFAs and their derivatives, the LC-PUFAs. The fatty acids analyzed and their retention times according to the purified standard are presented in Table 3.
The most abundant fatty acid was oleic acid (C18: 1n9c), which showed no differences across the different milk production phases;    et al., 2011). This may be due to differences in diet since these fatty acids and their precursors come from the diet.

| Trans fatty acid content in breast milk
The study looked for three TFAs-elaidic, linoelaidic, and petroselaidic fatty acids-but found only elaidic and linoelaidic acids in studied samples. There are few studies that analyze industrially produced TFAs in human milk, and even fewer that study the presence of these fatty acids in all three stages of lactation. However, the findings of the present study coincide with some previously reported works, in which the industrially produced TFA was found in the highest concentrations of iselaidic acid, followed by li-  (Daud et al., 2013). It should be noted that the common literature indicates that the most prevalent TFA in breast milk is elaidic acid, which is also the most prevalent industrially produced TFA in food (Hauff & Vetter, 2009;Krešić et al., 2013;De Souza et al., 2015;Villalpando, 2007).
The amounts found are also comparable with the values of elaidic acid found in colostrum (1.91%) and mature milk ( For comparative purposes, in addition to determining the content of TFAs C18:1t in breast milk using the methodology described above, the Craig-Schmidt formula was applied using the values of the estimated maternal intake of TFAs. In 1984, Craig-Schmidt published a work suggesting an equation for estimating the quantity of TFAs in breast milk based on the estimated dietary intake (Craig-Schmidt et al., 1984). The use of this formula has been extended to some current studies. We found that although the elaidic acid content of colostrum estimated using the Craig-Schmidt formula was higher than our own finding, it did not differ significantly (1.75% vs.

| Fatty acid content in breast milk, according to anthropometric measurements of the mother
The content of TFAs and EFAs in breast milk (colostrum, transitional milk, and mature milk) was compared against anthropometric measurements, according to WHO criteria (Organización Mundial de la Salud, 2000). The mothers were classified into two groups according to their BMI: Group 1: low and normal weight (BMI ≤24.9) and Group 2: overweight and obese (BMI ≥25.0). Mothers were also stratified according to their percentage of body fat: Group 1, decreased and healthy (≤32.99%) and Group 2, high and very high (≥33.0) (Gallagher et al., 2000). The anthropometric measurements of the mother (BMI and percentage of body fat) were not found to correlate with the composition of TFAs or EFAs in breast milk in any of the three stages.
This is comparable to another study in which there was also no re-

| Relationship between maternal intake and the content of trans fatty acids and essential fatty acids in milk
The results do not show a correlation between the content of TFAs in milk and the intake of TFAs, either expressed in grams or expressed as a percentage of the total caloric value of the diet obtained from the analysis of the 24HR recall (p > .05). Nor was the content of TFAs in milk found to correlate with the percentage of lipids, proteins, and carbohydrates consumed in the diet or with energy consumption.
These results coincide with those reported by Samur et al. (2009), who studied the composition of TFAs and its association with the diet of lactating women in Turkey also obtained using the 24HR. Although they found that there was no significant correlation between TFAs in milk and diet, they reported that mothers who presented a high level of trans isomers in their milk consumed significantly higher amounts of products rich in TFAs. There are other studies which have suggested a relationship between the TFA content in breast milk and TFA consumption by the mother: although Gomez Cortés and De la Fuente (Gómez-Cortés & De La Fuente, 2017) did not directly study mothers' diet, they studied and compared the composition of milk produced by rural and urban women, finding that the percentage of elaidic acid was significantly higher in women in urban areas (0.07% vs. 0.14%, respectively). Their assumption was that these women consume more TFAs because they had more access to industrialized food. A recent study by Perrin et al. (2018)  These results indicate that there are aspects of a mother's diet that may correlate with the content of fatty acids in breast milk, and that this correlation may be significant. This last aspect merits further study.

| Relationship between the content of essential fatty acids and trans fatty acids in colostrum, transitional milk, and mature milk
The correlation between EFAs and TFAs was evaluated in breast milk ( Table 5). In colostrum, among the n−6 series fatty acids, linoleic acid showed an inverse correlation with elaidic acid content and with total TFA content (p < .05); while AA, γ-linolenic acid, and LCPUFA n−6 (γ-linolenic acid + arachidonic acid) showed a positive correlation with both elaidic acid and total TFAs. Among the n−3 series fatty acids, linolenic acid and total n−3 series fatty acids correlated positively with elaidic acid content and total TFA content (p < .05). However, there was an inverse correlation between DHA content and both elaidic acid content and total TFAs. In the case of transitional milk, linoleic acid, AA, and total n−6 were negatively associated with linoelaidic acid (p < .05). However, with the exception of the EPA fatty acid, the n−3 family: linolenic acid, DHA, the sum of LC-PUFA n−3 (EPA + DHA) and total n−3, showed a positive correlation with the levels of elaidic acid, linoelaidic acid, and total of TFAs (p < .05). Finally, in mature milk, among the n−6 series fatty acids, linoleic acid showed a negative correlation with total TFAs and AA also a negative correlation with the content of elaidic and linoelaidic acid; while γ-linolenic acid and LC-PUFA n−6 had a positive correlation: γ-linolenic acid with linoelaidic acid and the total of TFAs, and LC-PUFA n−6 with linoelaidic acid content (p < .05). Furthermore, among the n−3 series fatty acids, linolenic acid, EPA, LC-PUFA n−3, and total n−3 showed a positive correlation with the levels of linoelaidic acid and total TFAs; the total of n−3 also showed a positive correlation with elaidic acid content (p < .05) ( Table 5).
Similar correlations were found between linolenic acid and total fatty acids of the n−3 series, because linolenic acid is the most prevalent fatty acid of the n−3 family. There were also similarities between the behavior of elaidic acid and the sum of total TFAs, since elaidic acid makes up 87.2% of total TFAs.
The few studies that exist on the correlation between TFAs and EFAs in mature milk do not coincide. Krešić et al. (2013) observed an inverse relationship between linoleic acid, linolenic fatty acids, EPA, DHA, and total LC-PUFA, with total TFAs in mature milk. De Souza Santos da Costa et al. (2016), on the other hand, looked for a correlation between the most abundant TFAs, elaidic acid, and concentrations of LC-PUFAs in mature milk, but were unable to find one. Samur et al., 2009(Samur et al., 2009) failed to observe a correlation between total elaidic acid or total TFAs and linoleic, linolenic, total n−3, and total n−6 fatty acids.
In our study, we did find a correlation between TFAs and EFA content in all three stages of maternal milk, although this correlation was, on many occasions, positive. In colostrum, despite the fact that linolenic acid was positively associated with elaidic acid content, the LC-PUFA of greater importance to the newborn's visual and nervous system development, DHA, was inversely related. This could be due to the negative effect of the presence of TFAs on the metabolism of EFAs, since TFAs compete for the same enzymes (∆5 and ∆6 desaturases), blocking them and preventing DHA biosynthesis from taking place in the mother's body, which can change the composition of breast milk (Krešić et al., 2013;Kummerow, 2009;Villalpando, 2007  The tables showing the TFA composition of food consumed frequently in various regions-including Mexico-must be kept continually up to date. For the moment, these tables afford a limited approach and may contribute to the underestimation of the consumption of TFAs as well as the association between TFAs in breast milk and TFAs in the mothers' diets. Likewise, a validated tool is required for collecting information on the intake of trans fats, which would allow for a more accurate estimate of their consumption.

| CON CLUS ION
In this study, TFAs were found in maternal milk. Olvera. We are also grateful to Marissa Nallely Nieto Escorcia for her support in handling and analyzing the samples.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L CO N S I D ER ATI O N S
This work was carried out in keeping with the ethical principles for medical research involving human subjects adopted in the 18th World Medical Assembly, Helsinki, Finland, June 1964. The study was approved by the Bioethics Committee of the School of Natural Sciences, UAQ. The study does not involve any risk, since the expression of breast milk is a common procedure. Participants included in the study received an evaluation of their body composition and a personalized dietary plan at the end of their participation in the study, as well as advice on breastfeeding beginning with the first meeting.

I N FO R M ED CO N S ENT
The women were invited to participate and received an explanation of the study. Those who met the inclusion criteria and accepted were given an informed consent form to sign.